Proteins A and G are often employed to purify antibodies by affinity chromatography. See e.g. R. Vola et al. (1994), Cell Biophys. 24-25: 27-36; Aybay and Imir (2000), J. Immunol. Methods 233(1-2): 77-81; Ford et al. (2001), J. Chromatogr. B 754: 427-435. These proteins are useful because they bind to a constant (FC) portion of many different antibodies. Recombinant fusion proteins including an FC portion of an IgG antibody can be purified using similar methods.
When a protein is produced for pharmacological use, it is important to remove toxic or immunogenic contaminants, such as other proteins. Specifically, Protein A is immunogenic and, in large amounts, potentially toxic. Some Protein A can leach into a sample during affinity chromatography when Protein A affixed to a solid support is used as an adsorbent. See e.g. Bensinger et al. (1984), J. Biol. Response Modif. 3: 347-351; Ventura et al. (1987), Cancer Treat. Rep. 71(4): 411-413 (both of which are incorporated herein in their entirety).
Bispecific monoclonal antibodies have been separated from other antibodies by binding and eluting them from hydroxyapatite subsequent to a pre-purification by affinity chromatography using Protein A affixed to a solid support as an adsorbent. Tarditi et al. (1992), J. Chromatography 599: 13-20; Ford et al. (2001), J. Chromatography B 754: 427-435. Monoclonal antibodies have also been bound and eluted from hydroxyapatite. Ahmad (1999), Ceramic Hydroxyapatite as a Polishing Step for Monoclonal Antibody Purification, Abstract from the Recovery of Biological Products Meeting #9 at Whistler, British Columbia.
The present invention provides a simplified and broadly applicable process for using hydroxyapatite chromatography for purification of antibodies and other proteins.